PROJECT SUMMARY Two-photon excitation (2PE) microscopy is a valuable tool for imaging thick tissues due to a deep penetration depth of near-infrared light and a good optical sectioning capability. In particular, the advent of genetically encoded fluorescent proteins allows us to exploit 2PE microscopy for studying structure, function and dynamics of subcellular components in living samples. By itself, 2PE microscopy has a somewhat lower spatial resolution than that of confocal microscopy but this has been overcome by combining 2PE with stimulated emission depletion (STED) microscopy. For example, 2PE-STED enabled us to investigate the neuronal connection or the neuronal activity upon stimulation, which significantly enhanced our understanding of the brain with superior spatial resolution. However, current 2PE-STED microscopy does not provide the capability of simultaneous imaging of multiple targets, which is critical to study interactions and dynamics of a variety of biomolecules. Conventional 2PE microscopy uses a mode-locked Ti:Sapphire laser system as a standard light source but this is generally bulky and is the primary cost driver of 2PE microscopy. For this reason, simultaneous three- or four-color 2PE imaging has been highly challenging, typically requiring at least two Ti:Sapphire lasers. Recently, many laser sources have advanced in high-power femtosecond pulse generation but their usable wavelength range, power or pulse repetition rate is not ideal for imaging tissues expressing fluorescent proteins. We propose to develop multicolor two-photon excitation STED microscopy with a multi-wavelength laser system integrated with multiple semiconductor lasers. This will bring several desirable features: (i) the semiconductor laser is much more affordable than a conventional system, which allows implementation of multiple lasers, (ii) the high-repetition-rate and lower peak power will reduce photobleaching and photodamage while maintaining high signal-to-noise ratio, (iii) the laser can be made to operate at many different wavelengths, and (iv) this ultrafast source is also ultracompact and it is possible to integrate into a miniaturized microscope. In order to achieve these goals, we will develop semiconductor- based laser light sources that will generate optical pulses with 1 ps pulse duration and >2 kW peak power at 920 nm and 850 nm wavelengths with a variable 0.3-1 GHz repetition rate. We will integrate this laser with a microscope to establish the 2PE-STED system. We propose the following Specific Aims: In Aim 1, we will develop multichannel semiconductor laser system. In aim 2, we will demonstrate 2PE imaging and characterize optical responses of fluorescent proteins. In aim 3, we will demonstrate multicolor 2PE-STED microscopy using the multichannel semiconductor laser system. Our proposal promises to provide a powerful deep-tissue imaging method with multicolor sub-diffraction resolution. Our affordable technique will be highly useful for application in many research areas including neuroscience and immunology.